Jinmai Jiang

Expression profiling identifies microRNA signature in pancreatic cancer

microRNAs are functional, 22 nt, noncoding RNAs that negatively regulate gene expression. Disturbance of microRNA expression may play a role in the initiation and progression of certain diseases. A microRNA expression signature has been identified that is associated with pancreatic cancer. This has been accomplished with the application of real‐time PCR profiling of over 200 microRNA precursors on specimens of human pancreatic adenocarcinoma, paired benign tissue, normal pancreas, chronic pancreatitis and nine pancreatic cancer cell lines. Hierarchical clustering was able to distinguish tumor from normal pancreas, pancreatitis and cell lines. The PAM algorithm correctly classified 28 of 28 tumors, 6 of 6 normal pancreas and 11 of 15 adjacent benign tissues. One hundred microRNA precursors were aberrantly expressed in pancreatic cancer or desmoplasia (p < 0.01), including microRNAs previously reported as differentially expressed in other human cancers (miR‐155, miR‐21, miR‐221 and miR‐222) as well as those not previously reported in cancer (miR‐376a and miR‐301). Most of the top aberrantly expressed miRNAs displayed increased expression in the tumor. Expression of the active, mature microRNA was validated using a real‐time PCR assay to quantify the mature microRNA and Northern blotting. Reverse transcription in situ PCR showed that three of the top differentially expressed miRNAs (miR‐221, ‐376a and ‐301) were localized to tumor cells and not to stroma or normal acini or ducts. Aberrant microRNA expression may offer new clues to pancreatic tumorigenesis and may provide diagnostic biomarkers for pancreatic adenocarcinoma.

Association of MicroRNA Expression in Hepatocellular Carcinomas with Hepatitis Infection, Cirrhosis, and Patient Survival

Purpose: MicroRNA (miRNA) is a new class of small, noncoding RNA. The purpose of this study was to determine if miRNAs are differentially expressed in hepatocellular carcinoma (HCC). Experimental Design: More than 200 precursor and mature miRNAs were profiled by real-time PCR in 43 and 28 pairs of HCC and adjacent benign liver, respectively, and in normal liver specimens. Results: Several miRNAs including miR-199a, miR-21, and miR-301 were differentially expressed in the tumor compared with adjacent benign liver. A large number of mature and precursor miRNAs were up-regulated in the adjacent benign liver specimens that were both cirrhotic and hepatitis-positive compared with the uninfected, noncirrhotic specimens (P < 0.01). Interestingly, all of the miRNAs in this comparison had increased expression and none were decreased. The expression of 95 randomly selected mRNAs was not significantly altered in the cirrhotic and hepatitis-positive specimens, suggesting a preferential increase in the transcription of miRNA. Comparing the miRNA expression in the HCC tumors with patients survival time revealed two groups of patients; those with predominantly lower miRNA expression and poor survival and those with predominantly higher miRNA expression and good survival (P < 0.05). A set of 19 miRNAs significantly correlated with disease outcome. A number of biological processes including cell division, mitosis, and G1-S transition were predicted to be targets of the 19 miRNAs in this group. Conclusion: We show that a global increase in the transcription of miRNA genes occurs in cirrhotic and hepatitis-positive livers and that miRNA expression may prognosticate disease outcome in HCC.

The Human Angiotensin II Type 1 Receptor +1166 A/C Polymorphism Attenuates MicroRNA-155 Binding

The adverse effects of angiotensin II (Ang II) are primarily mediated through the Ang II type 1 receptor (AT1R). A silent polymorphism (+1166 A/C) in the human AT1R gene has been associated with cardiovascular disease, possibly as a result of enhanced AT1R activity. Because this polymorphism occurs in the 3′-untranslated region of the human AT1R gene, the biological importance of this mutation has always been questionable. Computer alignment demonstrated that the +1166 A/C polymorphism occurred in a cis-regulatory site, which is recognized by a specific microRNA (miRNA), miR-155. miRNAs are noncoding RNAs that silence gene expression by base-pairing with complementary sequences in the 3′-untranslated region of target RNAs. When the +1166 C-allele is present, base-pairing complementarity is interrupted, and the ability of miR-155 to interact with the cis-regulatory site is decreased. As a result, miR-155 no longer attenuates translation as efficiently as demonstrated by luciferase reporter and Ang II radioreceptor binding assays. In situ hybridization experiments demonstrated that mature miR-155 is abundantly expressed in the same cell types as the AT1R (e.g. endothelial and vascular smooth muscle). Finally, when human primary vascular smooth muscle cells were transfected with an antisense miR-155 inhibitor, endogenous human AT1R expression and Ang II-induced ERK1/2 activation were significantly increased. Taken together, our study demonstrates that the AT1R and miR-155 are co-expressed and that miR-155 translationally represses the expression of AT1R in vivo. Therefore, our study provides the first feasible biochemical mechanism by which the +1166 A/C polymorphism can lead to increased AT1R densities and possibly cardiovascular disease.

The neuronal microRNA miR-326 acts in a feedback loop with Notch and has therapeutic potential against brain tumors

Little is known of microRNA interactions with cellular pathways. Few reports have associated microRNAs with the Notch pathway, which plays key roles in nervous system development and in brain tumors. We previously implicated the Notch pathway in gliomas, the most common and aggressive brain tumors. While investigating Notch mediators, we noted microRNA-326 was upregulated following Notch-1 knockdown. This neuronally expressed microRNA was not only suppressed by Notch but also inhibited Notch proteins and activity, indicating a feedback loop. MicroRNA-326 was downregulated in gliomas via decreased expression of its host gene. Transfection of microRNA-326 into both established and stem cell-like glioma lines was cytotoxic, and rescue was obtained with Notch restoration. Furthermore, miR-326 transfection reduced glioma cell tumorigenicity in vivo. Additionally, we found microRNA-326 partially mediated the toxic effects of Notch knockdown. This work demonstrates a microRNA-326/Notch axis, shedding light on the biology of Notch and suggesting microRNA-326 delivery as a therapy.

Human chromosome 21-derived miRNAs are overexpressed in down syndrome brains and hearts.

Down syndrome (DS), or Trisomy 21, is the most common genetic cause of cognitive impairment and congenital heart defects in the human population. To date, the contribution of microRNAs (miRNAs) in DS has not been investigated. Bioinformatic analyses demonstrate that human chromosome 21 (Hsa21) harbors five miRNA genes; miR-99a, let-7c, miR-125b-2, miR-155, and miR-802. MiRNA expression profiling, miRNA RT-PCR, and miRNA in situ hybridization experiments demonstrate that these miRNAs are overexpressed in fetal brain and heart specimens from individuals with DS when compared with age- and sex-matched controls. We hypothesize that trisomic 21 gene dosage overexpression of Hsa21-derived miRNAs results in the decreased expression of specific target proteins and contribute, in part, to features of the neuronal and cardiac DS phenotype. Importantly, Hsa21-derived miRNAs may provide novel therapeutic targets in the treatment of individuals with DS.

miR-132 and miR-212 are increased in pancreatic cancer and target the retinoblastoma tumor suppressor

Numerous microRNAs (miRNAs) are reported as differentially expressed in cancer, however the consequence of miRNA deregulation in cancer is unknown for many miRNAs. We report that two miRNAs located on chromosome 17p13, miR-132 and miR-212, are over-expressed in pancreatic adenocarcinoma (PDAC) tissues. Both miRNAs are predicted to target the retinoblastoma tumor suppressor, Rb1. Validation of this interaction was confirmed by luciferase reporter assay and western blot in a pancreatic cancer cell line transfected with pre-miR-212 and pre-miR-132 oligos. Cell proliferation was enhanced in Panc-1 cells transfected with pre-miR-132/-212 oligos. Conversely, antisense oligos to miR-132/-212 reduced cell proliferation and caused a G(2)/M cell cycle arrest. The mRNA of a number of E2F transcriptional targets were increased in cells over expressing miR-132/-212. Exposing Panc-1 cells to the β2 adrenergic receptor agonist, terbutaline, increased the miR-132 and miR-212 expression by 2- to 4-fold. We report that over-expression of miR-132 and miR-212 result in reduced pRb protein in pancreatic cancer cells and that the increase in cell proliferation from over-expression of these miRNAs is likely due to increased expression of several E2F target genes. The β2 adrenergic pathway may play an important role in this novel mechanism.

miR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines

Previous work by us and others reported decreased expression of miR-199a-3p in hepatocellular carcinoma (HCC) tissues compared to adjacent benign tissue. We report here a significant reduction of miR-199a-3p expression in 7 HCC cell lines. To determine if miR-199a-3p has a tumor suppressive role, pre-miR-199a-3p oligonucleotides were transfected into the HCC cell lines. Pre-miR-199a-3p oligonucleotide reduced cell proliferation by approximately 60% compared to control oligonucleotide in only two cell lines (SNU449 and SNU423); the proliferation of the other 5 treated cell lines was similar to control oligonucleotide. A pre-miR-199a-3p oligonucleotide formulated with chemical modifications to enhance stability while preserving processing, reduced cell proliferation in SNU449 and SNU423 to the same extent as the commercially available pre-miR-199a-3p oligonucleotide. Furthermore, only the duplex miR-199a-3p oligonucleotide, and not the guide strand alone, was effective at reducing cell viability. Since a CD44 variant was essential for c-Met signaling [V. Orian-Rousseau, L. Chen, J.P. Sleeman, P. Herrlich, H. Ponta, CD44 is required for two consecutive steps in HGF/c-Met signaling, Genes Dev. 16 (2002) 3074-3086] and c-Met is a known miR-199a-3p target, we hypothesized that miR-199a-3p may also target CD44. Immunoblotting confirmed that only the two HCC lines that were sensitive to the effects of pre-miR-199a-3p were CD44+. Direct targeting of CD44 by miR-199a-3p was confirmed using luciferase reporter assays and immunoblotting. Transfection of miR-199a-3p into SNU449 cells reduced in vitro invasion and sensitized the cells to doxorubicin; both effects were enhanced when hyaluronic acid (HA) was added to the cell cultures. An inverse correlation between the expression of miR-199a-3p and CD44 protein was noted in primary HCC specimens. The ability of miR-199a-3p to selectively kill CD44+ HCC may be a useful targeted therapy for CD44+ HCC.

Increased expression of microRNA-155 in Epstein-Barr virus transformed lymphoblastoid cell lines.

In a recent issue of Genes, Chromosomes & Cancer, Metzler et al. identified that the precursor to microRNA-155 (miR-155) was located within exon 3 of the noncoding RNA BIC (Metzler et al., 2004). In a prior issue of Genes, Chromosomes & Cancer, BIC was reported to be expressed at very high levels in Hodgkin lymphoma cell lines compared to nonHodgkin lymphoma cell lines (van den Berg et al., 2003). Increased BIC RNA was found in the lymphoblastoid cell lines JY25 and CB33 (Eis et al., 2005) and to a lesser extent in POP and RAY (van den Berg et al., 2003). The expression of miR-155 precursor was increased by 100-fold in children with Burkitt lymphoma compared to normal blood and pediatric leukemia patients (Metzler et al., 2004). Increased mature miR-155 was reported in cell lines and patient specimens of diffuse large Bcell lymphoma (DCBCL) and Hodgkin lymphoma (Eis et al., 2005). BIC was originally identified as a transcriptionally activated gene by promoter insertion at a common retroviral integration site in B-cell lymphomas induced by the avian leukemia virus (Tam et al., 1997). Identification of miR-155 within the BIC gene (Metzler et al., 2004) and subsequent expression of mature miR-155 in patient samples and cell lines (Eis et al., 2005) strengthens the case that the functional oncogenic product of BIC is the 21 nucleotide mature miR-155. This is supported by other evidence including that the miR-155 precursor is located within the highly conserved region of BIC (Tam, 2001). Differential miRNA expression has been shown in a number of human cancers (Calin et al., 2002; Michael et al., 2003; Takamizawa et al., 2004; Johnson et al., 2005); however the expression of most of these miRNAs are downregulated. miR-155 is one of the few miRNAs whose expression is increased in cancer. miRNAs are believed to function by binding to the 30 untranslated region of protein coding mRNAs by partial complimentarity, resulting in reduced translation of the target gene. We have recently developed a high throughput, real-time PCR assay to profile the expression of human miRNA precursors (Schmittgen et al., 2004; Jiang et al., 2005). The assay was used to profile the expression of 222 miRNA precursors in CD19þ B cells from three donors and in the lymphoblastoid cell line Wa C3 CD5þ. Wa C3 CD5þ was derived by immortalizing peripheral blood lymphocytes from a B-CLL patient by using Epstein-Barr virus, S. aureus Cowan 1, MP6-thioredoxin, and interleukin-2 (Wendel-Hansen et al., 1994). The expression of most of the miRNA precursors was comparable in CD19þ B cells and Wa C3 CD5þ (Fig. 1). The expression of miR-155 precursor was markedly up-regulated in Wa C3 CD5þ, representing a 110-fold higher expression in Wa C3 CD5þ compared to CD19þ B cells (Fig. 1). We were intrigued by the study of Küppers et al. (2003), who performed gene expression array analysis on various hematopoietic cancer cell lines including Hodgkin lymphoma, DLBCL, (GC and ABC types), lymphoblastoid cell lines, and Burkitt lymphoma (Küppers et al., 2003). The data were clustered into two groups based upon similarities to the expression profile of the ABC or GC phenotypes. The ABC-like cluster contained Hodgkin lymphoma (L-428, L-1236, KMH2, and HDLM2), DLBCL (Ly3 and Ly10), and EBV-transformed lymphoblastoid cell lines (Daikiki, NC6, CB33, RD, and IARC304) (Küppers et al., 2003). The GC cluster contained twelve cell lines including GC DLBCL (Ly1, Ly7, and Val) and Burkitt lymphoma lines Ramos, EB3, and Namalwa (Küppers et al., 2003). Interestingly, all of the cell lines with known miR-155 overexpression (L-428, L-1236, KM-H2, HDLM-2, Ly3, and Ly10 (Eis et al., 2005)) were present only in the ABC-like cluster, while those with basal miR-155 expression (Ramos, Ly8 and Ly1 (Eis et al., 2005)) were present in the GC cluster (Küppers et al., 2003). Since miR-155 was increased in Wa C3 CD5þ cells (Fig. 1) and BIC was increased in JY25 and CB33 (Eis et al., 2005), we hypothesized that miR-155 may be overexpressed in the EBV trans-

High-throughput real-time PCR.

Real-time PCR is presently the gold standard of gene expression quantification. Configuration of real-time PCR instruments with 384-well reaction blocks, enables the instrument to be used essentially as a low-density array. While PCR will never rival the throughput of microchip arrays, in situations where one is interested in assaying several hundreds of genes, high throughput, real-time PCR is an excellent alternative to microchip arrays. By combining SYBR green detection and 5 microL reaction volume, the associated costs of high-throughput real-time PCR are comparable to microarrays. Described here is a complete protocol to perform real-time PCR in a 384-well configuration. Examples are provided to access numerous PCR primer sequences that may be used for high-throughput real-time PCR. Methods of analysis are described to present real-time PCR data as heat maps and clustered similar to the presentation of cDNA microarray data. An example is provided to profile the expression of over 200 microRNA precursors using high-throughput real-time PCR.

MicroRNAs are mediators of androgen action in prostate and muscle.

Androgen receptor (AR) function is critical for the development of male reproductive organs, muscle, bone and other tissues. Functionally impaired AR results in androgen insensitivity syndrome (AIS). The interaction between AR and microRNA (miR) signaling pathways was examined to understand the role of miRs in AR function. Reduction of androgen levels in Sprague-Dawley rats by castration inhibited the expression of a large set of miRs in prostate and muscle, which was reversed by treatment of castrated rats with 3 mg/day dihydrotestosterone (DHT) or selective androgen receptor modulators. Knockout of the miR processing enzyme, DICER, in LNCaP prostate cancer cells or tissue specifically in mice inhibited AR function leading to AIS. Since the only function of miRs is to bind to 3′ UTR and inhibit translation of target genes, androgens might induce miRs to inhibit repressors of AR function. In concordance, knock-down of DICER in LNCaP cells and in tissues in mice induced the expression of corepressors, NCoR and SMRT. These studies demonstrate a feedback loop between miRs, corepressors and AR and the imperative role of miRs in AR function in non-cancerous androgen-responsive tissues.